Hepatitis B virus and hepatitis D virus infection in women with or at risk for HIV infection in the United States

Hepatitis D virus (HDV) requires co-infection with hepatitis B virus (HBV). Human immunodeficiency virus (HIV) shares transmission routes with these viruses. Among 4,932 US women infected with or at-risk for HIV during 1994–2015, HBV surface antigen (HBsAg) positivity was more common in women with HIV (2.8% vs. 1.2%; p = 0.001); HDV was more common among participants enrolled during 2013–2015 (p = 0.0004) and those with resolved rather than active hepatitis C (1.9% vs. 0.5%; p = 0.02). Among HBsAg-positive women (n = 117), HDV antibody prevalence was 22% and did not vary by HIV status; HDV infection was associated with the presence of advanced fibrosis/cirrhosis at enrollment (adjusted odds ratio, 5.70; 95% confidence interval, 1.46–22.29). Our results demonstrate the importance of HDV testing in HBV-infected US women.


Background
Hepatitis D virus (HDV) is a defective subviral pathogen that requires hepatitis B virus (HBV) for replication. An estimated 357,000 people in the United States are believed to have had past or ongoing HDV infection, although prevalence data are limited due to a paucity of previous studies (1).
HBV/HDV coinfected individuals experience more rapid progression to liver cirrhosis, hepatic decompensation, hepatocellular carcinoma, and death compared to those infected with HBV alone (2). HBV and HDV can be transmitted both sexually and through parenteral exposure. Given shared routes of transmission with human immunodeficiency virus (HIV) infection, people living with HIV (PLWH) or at risk for HIV infection are at higher risk for acquiring HDV. Furthermore, HDV-related liver disease may progress faster in PLWH, significantly impacting quality of life and survival (3). However, little is known about the prevalence of HDV infection in this population in the United States (4). With new, effective treatments for HDV potentially on the horizon (5,6), it is important to determine HDV prevalence in high-risk populations, such as PLWH or at risk for acquiring HIV infection, to inform HDV screening and treatment.
The Women's Interagency HIV Study (WIHS) is a large prospective study designed to investigate the treatment and prevention of HIV infection among US women. Using the HDV quantitative microarray antibody capture (Q-MAC) assay, we examined the prevalence of HDV infection among the WIHS participants overall and in subgroups defined by demographic, behavioral and clinical characteristics, including HIV infection status.

Study population
WIHS is a multi-center cohort study that was established in 1994 to investigate the natural history and treatment of HIV infection and associated morbidities among women living in the United States. Details regarding study methods and follow-up protocols have been previously described (7). WIHS investigators recruited 3,677 HIV-seropositive women and 1,305 sociodemographically similar HIV-seronegative women at 10 U.S. study sites over 4 enrollment waves between 1994 and 2015. Data are collected prospectively through semiannual physical examinations, biological specimen collections and structured interviewer-administered questionnaires to obtain information regarding sociodemographic and risk behaviors including whether the participant had ever injected drugs or participated in transactional sex (defined as exchange of sex for drugs, money, or shelter). In 2019, the WIHS became part of the MACS/ WIHS Combined Cohort Study (MWCCS) with most women continuing enrollment under a similar protocol (8).

Laboratory methods
Testing was performed on blood specimens collected at "baseline" (study entry for 99.8% of participants). As previously described (9), assays from Abbott Laboratories were used to detect hepatitis B surface antigen (HBsAg; Auszyme Microparticle enzyme immunoassay [EIA]). We considered women who tested positive for HBsAg at baseline to have "active HBV infection" at that time point. In these participants, we used HBsAg test results from subsequent visits, performed using the Seimens ADVIA Centaur Immunoassay System, and classified "chronically infected" participants as those with a second positive HBsAg result at least 180 days after the initial positive result.
Hepatitis C (HCV) antibody status was assessed in baseline samples using enzyme immunoassay (EIA) 2.0 [Abbott Laboratories] and 3.0 [Ortho-Clinical Diagnostics] with testing for HCV RNA using either the COBAS Amplicor HCV Detection Kit, COBAS Taqman Assay [Roche Diagnostics] or Quantiplex 2.0 branched chain DNA-enhanced label amplification assay [Bayer-Versant Diagnostics] in participants who were positive for HCV antibody. We defined HCV status as "never infected" for participants who tested negative for HCV antibodies, "resolved infection" for those who tested positive for anti-HCV but negative for HCV RNA and "actively infected" for those who tested positive for HCV RNA. HIV infection status was based on results of an enzyme-linked immunosorbent assay with western blot confirmation [NASBA/NuciSens HIV RNA assay, BioMerieux] (10).
Because HDV replication requires HBsAg, we limited testing for HDV to reposited baseline samples from participants with active HBV infection. As previously described (12), HDV Q-MAC is a highthroughput assay with high sensitivity and specificity for detecting anti-HDV immunoglobulin G (12,13). After excluding two participants without an available specimen, we tested 117 HBsAgpositive participants for anti-HDV.

Statistical analysis
Among women who were tested for HBsAg, we examined the prevalence of active HBV infection and the prevalence of HDV infection. We also determined anti-HDV seroprevalence among participants with active HBV infection. To assess whether infection prevalence differed in subgroups, we examined prevalence in strata defined by demographic, behavioral and clinical characteristics. Prevalence estimates were compared by χ 2 test or Fisher's exact test (when ≥25% of cells had a count <5).
Unconditional logistic regression was used the assess the association between HDV infection and selected variables, including year of enrollment, transactional sex, HCV status, injection drug use, and HIV infection. Multivariable analyses were not possible due to sparse data for some of those variables.
Polytomous logistic regression models were used to estimate the odds ratio (OR) and 95% confidence interval (CI) for associations between HDV infection and the aspartate aminotransferase to platelet ratio index (APRI). Measures of APRI were categorized in the following manner: no significant fibrosis (<0.5), significant fibrosis (0.5 to <1.0), advanced fibrosis/cirrhosis (≥1.0). Age at baseline, HCV status (dichotomized as actively infected vs. never infected/resolved infection) and HIV status were considered as potential confounders.
All analyses were performed with SAS software version 9.4 (SAS Institute, Inc.)
We examined the prevalence of active HBV infection, by demographic, behavioral and clinical characteristics (Supplemental Table 1 Among WIHS participants overall, 26/4932 (0.5%) were positive for anti-HDV (Table 2). Anti-HDV prevalence did not differ by age, but participants who entered the cohort in 2013-2015 were more likely to be anti-HDV positive compared to those who enrolled in 1994-95 (13 [1.5%] vs. 10 [0.4%]; p = 0.0004). With the exception of one participant whose country of birth was missing, all anti-HDV seropositive participants were born in the United States. Women who reported injection drug use or transactional sex had a higher prevalence of anti-HDV seropositivity, although those differences were not statistically significant. Compared to the women who enrolled in 1994-1995, the participants who enrolled in 2013-2015 were less likely to report injection drug use (61 [ Among the 117 women with active HBV infection who were tested for anti-HDV, 26 (22.2%) were positive for HDV antibody ( Table 2). HDV prevalence increased with age at enrollment. While a test for trend in age categories was not significant, median age was 39.0 years for HDV-positive women and 34.0 years for HDV-negative women (p = 0.02). As per the overall cohort, among women with active HBV infection, anti-HDV seropositivity was significantly We examined the relationship between HDV infection, and the presence of fibrosis as defined by APRI-based categories among the participants with active HBV. Compared to women who had no significant fibrosis, those with advanced fibrosis/cirrhosis where much more likely to be positive for HDV, (OR = 6.00; 95%CI: 1.61-22.42) ( Table 3). After adjusting for age at baseline, this association was slightly attenuated (adjusted OR = 5.70; 95%CI: 1.46-22.29). HCV status did not confound this relationship. We were unable to assess potential confounding by HIV status because all participants with

Discussion
We evaluated the prevalence of anti-HDV seropositivity in a cohort of US women with or at risk of HIV infection. Using an assay with excellent performance characteristics for detecting anti-HDV, 22% of women with active HBV infection had evidence of HDV infection. While the women with HIV had a significantly higher prevalence of active HBV infection, anti-HDV prevalence did not differ by HIV status, although power was limited.
While anti-HDV seroprevalence was common among HBsAgpositive women enrolled in WIHS, the prevalence was lower than that reported in previous studies. Among people who inject drugs enrolled in the Urban Health Study (UHS), we found that 35.6% of those with active HBV infection were positive by the HDV QMAC assay (13). Other studies of HBV/HDV co-infection among individuals in the US who inject drugs reported prevalence estimates from 42 to 67% (14)(15)(16). In the 2011-2016 National Health and Nutrition Examination Survey, which is based on the general US population, investigators reported anti-HDV prevalence was 42% in those with active HBV. (1) Reported differences in anti-HDV prevalence may reflect differences in population, associated risk factors and test characteristics of anti-HDV assays. A limitation of this study is the lack of data on HDV RNA; nevertheless, in a previous study conducted in the United States by our group, all participants who tested positive by QMAC was also found to be positive for HDV RNA (13).
Anti-HDV seroprevalence was highest among the women who enrolled during 2013-2015, which is the latest period of enrollment. However, that finding should be interpreted with caution because WIHS enrollment in 2013-2015 was almost exclusively from southern U.S. states, whereas participants from other regions predominated in earlier enrollment waves. The higher prevalence of anti-HDV among the most recent WIHS enrollees thus could reflect either temporality or geography. To our knowledge, prevalence by geographical region has not been assessed in national surveys of HDV infection in the United States (1) and might be the subject of future studies.
Interactions between hepatitis viruses affect viral clearance (13). In the cohort overall, active HBV infection was more common in WIHS participants with resolved hepatitis C than those with active HCV infection, or those who had never been infected with HCV. These results correspond with our observations in UHS (13). In the cohort overall, anti-HDV prevalence was lower in those with active HCV infection than in those with resolved HCV infection. Among HBsAg+ positive participants, there were no meaningful difference in HDV prevalence by HCV status, whereas in UHS, we saw a slightly higher HDV prevalence in HBsAg+ participants with resolved compared to chronic HCV infection (13). Due to the cross-sectional design of these studies, temporality of viral acquisition could not be assessed, nevertheless, these results suggest that active HBV suppressed HCV infection in these populations. Longitudinal studies are needed to clarify interactions among hepatitis viruses.
In accordance with previous studies (2, 17), we found an association between HDV status and advanced fibrosis/cirrhosis at enrollment. Although we were limited by our lack of data on duration of infection, adjusting for age at baseline as a proxy measure still yielded a significant association between HDV status and advanced fibrosis/cirrhosis as defined by APRI.
Current guidelines for HDV testing recommend that persons belonging to certain 'high-risk' groups (including those with HIV or HCV infection, persons who have ever injected drugs, individuals with multiple sex partners) who test positive for HBsAg should also be tested for HDV infection (18). If we consider a history of transactional sex as an indicator of multiple sex partners, 89.7% of the women who were positive for HBsAg and 88.4% of the women who were positive for HDV had at least one risk factors that should prompt clinical testing for HDV. Data on previous HDV testing in clinical practice is not available for WIHS participants.
Our results provide additional evidence that HDV is common among individuals with active HBV infection in the US and support current recommendations that persons belonging to 'high-risk' groups who test positive for HBsAg should be tested for HDV infection (18). Little is known about the uptake of HDV screening recommendations in the United States. Future efforts should evaluate HDV testing rates in the US and seek to raise awareness regarding the importance of screening for this highly pathogenic virus.

Data availability statement
Access to individual-level data from the MACS/WIHS Combined Cohort Study Data (MWCCS) may be obtained upon review and approval of a MWCCS concept sheet. Links and instructions for online concept sheet submission are on the study website (https:// statepi.jhsph.edu/mwccs/work-with-us/).

Author contributions
IA, PM, RP, and TO'B contributed to the conception and design of the study. PL and JG conducted laboratory analyses. AA, MA, HB, AF, MP, JP, AR, AS, ES, and MK manage the cohort and corresponding data. IA conducted the statistical analyses and took the lead in writing the manuscript. All authors provided critical feedback and review of the manuscript. "no significant fibrosis", <0.5 (N = 84); "significant fibrosis", 0.5 to < 1.0 (N = 22); "advanced fibrosis/cirrhosis", ≥1.0 (N = 11). *value of ps calculated as p-trend.